1. Field of the Invention
The present invention relates to a biological support for cell cultures, constituted by a coagulated mixture of a concentrate of plasma proteins and thrombin, its use in the preparation of keratinocyte cultures and their transport in the form of reconstituted epidermises, and their use for therapeutic purposes.
2. Description of Related Art
The reconstitution in a laboratory of a living skin similar to the human skin from a few cells obtained from a biopsy, or of a simplified skin performing the physiological functions of a normal skin, is being studied extensively In the aim of replacing skin damaged by a serious disease (genetic, etc.) or destroyed by major burns.
The skin is a complex organ composed of three juxtaposed tissues: the epidermis, 85% of which is constituted by keratinocytes which form the impermeable horny layer that isolates the body from the outside environment; the dermis, which comprises cells, including fibrocytes, separated by a connective tissue composed mainly of collagen; the dermis lies on the hypoderma, which includes the cells dedicated to storing fats. Artificial reconstitution of such a complex organ thus poses numerous problems.
The first tissue to have been partially reconstituted in vitro was the dermis, this being achieved by the Bell team (Bell et al.) Proc. Natl. Aced. Sci. 76-1979-1274).
Starting with skin biopsies, fibroblasts have been successfully established in cultures, first in monolayers, then, after a number of passages, by dispersing these cells in culture medium containing collagen (extracted from rat's tail tendons), the latter forming a gel and permitting three-dimensional cultures. In such cultures, the fibroblasts can be seen interacting with the matrix of the collagen, organizing it and contracting It as in a normal dermis. This tissue, reconstituted in vitro, is known as an "equivalent dermis". After a few weeks' growth, the mechanical qualities of the equivalent dermis allow it to be used for grafting onto a patient or injured person. It does not appear to be rejected by its host. However, this equivalent dermis is merely a temporary dressing: it cannot restore the the cutaneous barrier function.
Furthermore, the Green team (H. Green et al. Proc. Natl. Aced. Sci. 76, 1979, 5665) has developed a method and a culture medium enabling keratinocytes to be grown for long periods. This method includes inoculating the keratinocytes dispersed with trypsin on a pre-established monolayer of fibroblasts, in particular 3T3 cells, lethally irradiated and which serve as a nutritive layer and as a matrix. The epidermal layer develops very rapidly to form a tissue having a thickness of 3 to 5 cells; it can be grafted onto a patient and continue to differentiate in situ. It has already proved possible to save those suffering from severe burns using this technique (G. Gallico et al. New England J. Med. 311, 1984, 448).
With Green's technique, it is possible to obtain, from a biopsy of two square centimeters an epidermis of one square meter in the space of three weeks.
Recovery of the reconstituted tissue in order to make a graft therefrom still poses a number of technical problems. It is, indeed, necessary to detach the cells from the culture dish, using an enzyme treatment, without dissociating them from one another; during this operation, one always observes a retraction of the cell layer, hence a loss of a certain percentage of the surface area of the graft. Once the reconstituted tissue has been detached, it has to be fastened to a support that enables it to be transported and grafted onto the patient. A vaseline treated gauze dressing is generally used. All these manipulations are delicate and time consuming.
It would thus be highly beneficial to have at one's disposal novel biological supports that can be resorbed in time by the patient who has received the graft and which simplify the handling of the cells. In addition, to ensure their availability, these supports or their constituents would have to lend themselves to preparation and packaging in accordance with industrial processes.